Innate immune cells constantly evaluate host mucosal surfaces and peripheral tissues for signs of infection or injury. The host must find a balance between tolerance of beneficial microorganisms and minor non-pathological microbial encounter vs. the development of a robust immune response to more serious infections. Emerging evidence suggests that this decision is made by the cell based on the strength and combination of signals it receives from its engagement with microorganisms and endogenous stimuli. These signals are sensed primarily by various classes of pattern recognition receptors (PRR), and while there has been remarkable progress in characterizing the individual signaling pathways induced by these receptors, relatively few studies have addressed how immune cells integrate combined PRR inputs and the combination of these signals with others arising from soluble host derived substances such as cytokines, lipids, and metabolites. We have previously analyzed the macrophage response to single and pairwise combinations of toll-like receptor (TLR) ligands, identifying characteristics of signaling pathway synergy and antagonism in signaling and cytokine outputs (Lin et al (2017) Cell Syst. 5: 25; Gottchalk et al (2016) Cell Syst. 2: 378). These studies emphasized the importance of feedback control in regulation of signaling flux, and in FY 2019 we have continued our investigation of this control phenomenon on two fronts. Firstly, our prior screening work suggested a negative regulatory role for the protein TANK in synergy between the Myd88 and TRIF branches of the TLR signaling response (Lin et al (2017) Cell Syst. 5: 25). Using primary macrophages from TANK-deficient mice, we now demonstrate that this negative regulatory function acts upstream of the primary MAPK and NF-kB signaling pathways and involves the atypical IkB kinase, IKKe. We are uncovering mechanistic aspects of this regulation and we also demonstrate a critical physiological role for this negative regulation in characterizing a pre-disposition to auto-inflammation in aging TANK and IKKe-deficient mice. Secondly, in collaboration with the Lymphocyte Biology Section of the LISB, a rigorous analysis of macrophages exposed to a matrix of increasing concentrations of paired TLR ligands has identified an acute negative feedback phenomenon that is selectively engaged during gram-negative bacterial infection. This anti-inflammatory feedback control is dependent on specific negative regulatory genes that are induced by type-I IFN, and has features that are independent of the established IL-10-dependent feedback loop. A manuscript describing this work was recently accepted for publication in eLife. The work above supports a critical role for type I IFN and interferon-stimulated genes (ISGs) in the host response to Gram-negative bacterial infection. To investigate this further, we have used the Gram-negative bacteria Burkholderia cenocepacia, which replicates in the host cell cytoplasm, to uncover a negative relationship between type I IFN signaling and the replicative potential of invading bacteria. Firstly, cells infected with these bacteria produce significant levels of IFN beta as well as ISGs. Cells pre-treated with IFN beta are less permissive to bacterial replication, while cells from mice lacking the type I IFN receptor (Ifnar1) have increased levels of bacterial replication, and higher amounts of cell death, compared to wild-type cells. Interestingly, this phenotype is type I IFN-specific, as pre-treatment with IFN-gamma has no effect on bacterial replication. In contrast, these phenotypes are reversed in cells infected with Salmonella, which replicates within in modified vacuole. This suggests that different IFN classes (and induced ISGs), may confer host protection against microbes replicating in different cellular niches. In FY 2019 we have characterized the host sensors responsible for type I IFN induction during Gram-negative bacterial infection and completed a broad transcriptomic profiling of bacterially infected WT and Ifnar1-deficient cells to identify the ISGs induced by different bacterial stimuli. This data is currently being prepared for publication. While the above studies highlight important immune regulatory autocrine effects of type I IFN in inflammatory macrophages, TLR activation also induces acute IFN-independent expression of the anti-inflammatory cytokine IL-10. In FY 2019, further insight to this process has been obtained from a characterization of the effect of the second messenger cAMP on LPS-induced IL-10 expression in macrophages. The cAMP pathway represents a critical immune regulatory mechanism through which host mediators such as eicosanoids regulate and limit inflammation. This effect has been shown in part to be mediated through cAMP-dependent enhancement of IL-10 mRNA expression, but the transcriptional control mechanisms are poorly defined. We show that cAMP can directly upregulate IL-10 expression and synergizes with LPS through a Myd88-dependent process. This study was published in Mediators of Inflammation. To further address how the TLR signaling network might mediate responses specific to combined TLR stimuli, we have investigated the localization dynamics of proximal TLR pathway components in response to single vs. combined ligands. In this context, we previously identified an IRAK1-containing complex that directly links multi-TLR signaling to inflammasome activation. IRAK1 containing bodies, that were distinct from myddosomes and trifosomes, were formed on co-stimulation of TLR4 and TLR1/2 or on bacterial infection. We found these complexes simultaneously recruited the inflammasome adaptor ASC, facilitating dual-TLR ligand-primed inflammasome activation that was diminished in Irak1-deficient macrophages. In FY 2019, we discovered an inflammasome licensing function for IRAK1 during dual-TLR priming, confirmed IRAK1-ASC association using a proximity ligation assay, and showed that this function of IRAK1 is required for IL-1 family cytokine induction during lung infection with Gram-negative bacteria. These data suggest a critical role for IRAK1-containing complexes in shaping the immune response to a multi-PAMP pathogen. This work will be submitted for publication in the near future. In FY 2019 we have also continued studies to investigate the cell signaling pre-requisites that link the priming and triggering events that control inflammasome activation and release of the IL-1/IL-18 family of cytokines. This pathway is frequently dysregulated in the inflammatory and metabolic diseases that place a substantial burden on global human health, and inflammasome regulation is closely linked to TLR activation during its priming phase. Moreover, several of our projects suggest unappreciated roles for TLR pathway components in the inflammasome response, independent of their role in transcriptional induction of inflammasome genes. We are developing a number of strategies and signaling reporter assays to determine the influence of cell signaling pathways and processes (many of them strongly influenced by cellular metabolic state) to the inflammasome response. This line of enquiry is ongoing.